PROJECT SUMMARY Tissue structure alterations are primary determinants of many developmental, physiological, and pathophysiological processes and often involve the coordinated movements of groups of physically interacting cells, a phenomenon referred to as collective cell migration. This phenomenon takes many different forms in a variety of processes such as embryonic development, wound healing, and cancer metastasis. Understanding the determinants and regulators of collective cell migration is therefore of great importance to human health. The critical processes mediating collective cell migration are thought to involve force generation and mechanical coupling among cells, but the underlying molecular mechanisms remain poorly understood. The long-term goal of this work is to understand the key mechanically-sensitive mechanisms mediating collective cell migration. Toward this goal, we have created and validated a set of innovative techniques for studying molecular scale, mechanically-sensitive processes within collectively migrating cells. Specifically, we focus on the mechanical linker protein vinculin, given its ability to regulate force-induced adhesion strengthening, established role in the development of load-bearing tissues, and emerging function as a mechanically-sensitive regulator of tumor progression. The overall objective of this proposal is to use these techniques to develop and test a novel conceptual model of collective cell migration in which forces generated by a leader cell activate vinculin-associated mechanosensitive pathways in surrounding cells to initiate coordinated directional migration. We will determine if 1) collectively migrating cells generate spatial gradients of molecular tension across vinculin, 2) spatial variations in force lead to the differential activation of mechanically sensitive signaling, 3) the relationship between vinculin load and vinculin dynamics is spatially organized and biochemically regulated during collective cell migration, and 4) cellular adhesion structure stability determines the form of collective cell migration. An enhanced mechanistic understanding of these processes would increase our fundamental knowledge of the regulation of tissue structure. Thus, these studies are relevant to the NIH's mission, as they will lead to new insights in many fields including cancer, birth defects, wound healing, and tissue regeneration.